Target (protein/gene name): Biotin Carboxylase NCBI #: Reference Sequence NC_002758.2 Protein ID (NP or XP #) or Wolbachia#: BAB57768 Organism (including strain): Methicillin-resistant Staphylococcus aureus, Mu50 Etiologic Risk Group (see link below): RG2
Disease Information (sort of like the Intro to your Mini Research Write up):
MRSA is a bacterial infection from Methicillin-resistant Staphylococcus aureus. Traditionally, S. aureus causes skin infections (cellulitis, impetigo), food poisoning, and bacteremia. Recently, strains of S. aureus have become resistant to common beta-lactam antibiotics, which include penicillins. This resistance does not make MRSA more virulent, but does make it substantially more difficult to treat. MRSA is especially dangerous in hospitals, where those with weakened immune systems are more prone to infection. Worldwide, up to 53 million people are thought to carry MRSA. The current treatment of MRSA are glycopeptide antibiotics such as vancomycin, however there are strains of S. aureus that have become resistant even to vancomycin (VISA). Link to TDR Targets page (if present): n/a Link to Gene Database page NCBI: https://www.ncbi.nlm.nih.gov/protein/BAB57768.1
Essentiality of this protein:
Bacterial acetyl-CoA carboxylase is an essential enzyme in bacteria because it catalyzes the first step in fatty acid synthesis. The enzyme produces malonyl-CoA, which is a building block for fatty acids. Fatty acids can then be incorporated into phospholipids, which form much of the lipid bilayer in the cell. Inhibition of this enzyme will deprive the bacteria of fatty acids, which can lead to cell death and inability to reproduce since the cell membrane cannot be replenished. Furthermore, products of this pathway are used in quorum sensing and protein modification. Is it a monomer or multimer as biological unit: dimer
Complex of proteins?:
Biotin carboxylase is part of bacterial acetyl-CoA carboxylase, an enzyme that contains three separate proteins: biotin carboxylase (BC), biotin carboxyl carrier protein (BCCD), and carboxyltransferase (CT). Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
Prototype inhibitor found for biotin carboxylase in Haemophilus influenzae *EC#: 6.3.4.14 Link to BRENDA EC# page: http://www.brenda-enzymes.org/enzyme.php?ecno=6.3.4.14 Enzyme Assay information: A continuous, spectrophotometric assay is available. Link to paper. Can we just measure the BC activity? - Maybe just use Biotin and not BCCP? - Dr. B 060617
BC only assay here, and another one -- links to assay reagents (substrates) pages. --- List cost and quantity of substrate reagents, supplier, and catalog #
Structure (PDB or Homology model) PDB #: 2VPQ Similarity to Human:
In eukaryotes, the BC, CT and BCCD subunits reside in a single polypeptide chain. In bacteria, ACC assembles as a multisubunit complex with each of the reactions performed by a separate enzyme. These differences in tertiary structure can provide for selective inhibitors.
Current Inhibitors: One weak inhibitor has been discovered. Expression Information (has it been expressed in bacterial cells): It can and has been expressed in E. coli. Expressed in cytoplasm. ACC is essential for growth, is expressed at all times, expression is also very regulated to maintain the correct stoichiometry of subunits. Purification Method: Standard nickel affinity column chromatography can be used. Image of protein (PyMol with features delineated and shown separately): *Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
Chain A Sequence:
MKKVLIANRGEIAVRIIRACRDLGIQTVAIYSEGDKDALHTQIADEAYCVGPTLSKDSYLNIPNILSIATSTGCDGVHPGYGFLAENADFAELCEACQLKFIGPSYQSIQKMGIKDVAKAEMIKANVPVVPGSDGLMKDVSEAKKIAKKIGYPVIIKATGGGGKGIRVARDEKELETGFRMTEQEA
QTAFGNGGLYMEKFIENFRHIEIQIVGDSYGNVIHLGERDCTIQRRMQKLVEEAPSPILDDETRREMGNAAVRAAKAVNYENAGTIEFIYDLNDNKFYFMEMNTRIQVEHPVTEMVTGIDLVKLQLQVAMGDVLPYKQEDIKLTGHAIEFRINAENPYKNFMPSPGKIEQYLAPGGYGVRIESA
CYTNYTIPPYYDSMVAKLIIHEPTRDEAIMAGIRALSEFVVLGIDTTIPFHIKLLNNDIFRSGKFNTNFLEQNSIMNDEG
Chain B Sequence:
MKKVLIANRGEIAVRIIRACRDLGIQTVAIYSEGDKDALHTQIADEAYCVGPTLSKDSYLNIPNILSIATSTGCDGVHPGYGFLAENADFAELCEACQLKFIGPSYQSIQKMGIKDVAKAEMIKANVPVVPGSDGLMKDVSEAKKIAKKIGYPVIIKATAGGGGKGIRVARDEKELETGFRMTEQE
AQTAFGNGGLYMEKFIENFRHIEIQIVGDSYGNVIHLGERDCTIQRRMQKLVEEAPSPILDDETRREMGNAAVRAAKAVNYENAGTIEFIYDLNDNKFYFMEMNTRIQVEHPVTEMVTGIDLVKLQLQVAMGDVLPYKQEDIKLTGHAIEFRINAENPYKNFMPSPGKIEQYLAPGGYGVRIES
ACYTNYTIPPYYDSMVAKLIIHEPTRDEAIMAGIRALSEFVVLGIDTTIPFHIKLLNNDIFRSGKFNTNFLEQNSIMNDEG Length of your protein in Amino Acids: 451 amino acids. Molecular Weight of your protein in kiloDaltons: 50.05 kDA Molar Extinction coefficient of your protein at 280 nm wavelength: 28685 mol^-1cm-1 TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). *CDS Gene Sequence (paste as text only): ATGAAAAAGGTTTTAATTGCAAACCGCGGTGAAATCGCAGTTAGGATTATTCGCGCTTGTCGTGATTTAGGCATCCAAACTGTTGCAATCTATTCTGAAGGGGATAAAGATGCGCTACATACTCAAATTGCTGATGAAGCATATTGCGTAGGTCCCACTTTGTCTAAAGATTCATATTTAAATATTCCGAACATCTTATCT ATTGCAACTTCTACAGGTTGTGATGGCGTCATCCGGGTTATGGCTTTTTAGCTGAAAATGCTGACTTTGCAGAATTATGCGAAGCATGCCAATTGAAGTTCATTGACCAAGTTATCAATCTATCCAAAAAATGGGTATCAAAGATGTTGCTAAGGCAGAAATGATCAAAGCCAATGTTCCAGTTGTTCCTGGTAGTGAC GGTTTAATGAAAGACGTCTCAGAAGCTAAGAAAATCGCTAAGAAAATTGGCTATCCGGTCATCATTAAAGCTACTGCTGGTGGTGGCGGAAAAGGTATCCGTGTTGCTCGTGATGAAAAAGAACTTGAAACTGGCTTCCGAATGACAGAACAAGAAGCTCAAACTGCATTTGGTAATGGTGGACTTTATATGGAGA AATTCATCGAAAACTTCCGCCATATTGAAATCCAAATTGTTGGGGACAGCTATGGTAATGTAATTCATTTAGGAGAACGTGATTGTACAATTCAAAGACGTATGCAGAAATTAGTGGAAGAAGCACCTTCCCCAATTTTAGATGATGAAACACGTCGTGAAATGGGAAATGCCGCAGTTCGTGCAGCGAAAGCTGTA AATTATGAAAATGCGGGAACAATTGAGTTTATATATGATTTAAATGATAATAAATTTTATTTTATGGAAATGAATACACGTATTCAAGTAGAACATCCTGTAACTGAAATGGTAACAGGAATTGATTTAGTTAAATTACAATTACAAGTTGCTATGGGTGACGTGTTACCGTATAAACAAGAAGATATTAAATAACAGGACA CGCAATTGAATTTAGAATTAATGCTGAAAATCCTTACAAGAACTTTATGCCATCACCAGGTAAAATTGAGCAATATCTTGCACCAGGTGGATATGGTGTTCGAATAGAGTCAGCATGTTATACTAATTATACGATACCGCCATATTATGATTCGATGGTAGCGAAATTAATCATACATGAACCGACACGAGATGAAGCGAT TATGGCTGGCATTCGTGCATTAAGTGAATTTGTGGTTCTTGGTATTGATACAACTATTCCATTCCATATTAAATTATTGAATAACGATATATTTAGAAGCGGTAAATTTAATACAAACTTTTTAGAGCAAAATAGCATTATGAATGATGAAGGTTAA
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
*GC% Content for gene:
output
*GC% Content for gene (codon optimized):
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences): **
NCBI #: Reference Sequence NC_002758.2
Protein ID (NP or XP #) or Wolbachia#: BAB57768
Organism (including strain): Methicillin-resistant Staphylococcus aureus, Mu50
Etiologic Risk Group (see link below): RG2
Disease Information (sort of like the Intro to your Mini Research Write up):
MRSA is a bacterial infection from Methicillin-resistant Staphylococcus aureus. Traditionally, S. aureus causes skin infections (cellulitis, impetigo), food poisoning, and bacteremia. Recently, strains of S. aureus have become resistant to common beta-lactam antibiotics, which include penicillins. This resistance does not make MRSA more virulent, but does make it substantially more difficult to treat. MRSA is especially dangerous in hospitals, where those with weakened immune systems are more prone to infection. Worldwide, up to 53 million people are thought to carry MRSA. The current treatment of MRSA are glycopeptide antibiotics such as vancomycin, however there are strains of S. aureus that have become resistant even to vancomycin (VISA).
Link to TDR Targets page (if present): n/a
Link to Gene Database page NCBI: https://www.ncbi.nlm.nih.gov/protein/BAB57768.1
Essentiality of this protein:
Bacterial acetyl-CoA carboxylase is an essential enzyme in bacteria because it catalyzes the first step in fatty acid synthesis. The enzyme produces malonyl-CoA, which is a building block for fatty acids. Fatty acids can then be incorporated into phospholipids, which form much of the lipid bilayer in the cell. Inhibition of this enzyme will deprive the bacteria of fatty acids, which can lead to cell death and inability to reproduce since the cell membrane cannot be replenished. Furthermore, products of this pathway are used in quorum sensing and protein modification.
Is it a monomer or multimer as biological unit: dimer
Complex of proteins?:
Biotin carboxylase is part of bacterial acetyl-CoA carboxylase, an enzyme that contains three separate proteins: biotin carboxylase (BC), biotin carboxyl carrier protein (BCCD), and carboxyltransferase (CT).
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
Prototype inhibitor found for biotin carboxylase in Haemophilus influenzae
*EC#: 6.3.4.14
Link to BRENDA EC# page: http://www.brenda-enzymes.org/enzyme.php?ecno=6.3.4.14
Enzyme Assay information:
A continuous, spectrophotometric assay is available. Link to paper.
Can we just measure the BC activity? - Maybe just use Biotin and not BCCP? - Dr. B 060617
BC only assay here, and another one
-- links to assay reagents (substrates) pages.
--- List cost and quantity of substrate reagents, supplier, and catalog #
Structure (PDB or Homology model)
PDB #: 2VPQ
Similarity to Human:
In eukaryotes, the BC, CT and BCCD subunits reside in a single polypeptide chain. In bacteria, ACC assembles as a multisubunit complex with each of the reactions performed by a separate enzyme. These differences in tertiary structure can provide for selective inhibitors.
Current Inhibitors:
One weak inhibitor has been discovered.
Expression Information (has it been expressed in bacterial cells): It can and has been expressed in E. coli. Expressed in cytoplasm. ACC is essential for growth, is expressed at all times, expression is also very regulated to maintain the correct stoichiometry of subunits.
Purification Method: Standard nickel affinity column chromatography can be used.
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
Chain A Sequence:
MKKVLIANRGEIAVRIIRACRDLGIQTVAIYSEGDKDALHTQIADEAYCVGPTLSKDSYLNIPNILSIATSTGCDGVHPGYGFLAENADFAELCEACQLKFIGPSYQSIQKMGIKDVAKAEMIKANVPVVPGSDGLMKDVSEAKKIAKKIGYPVIIKATGGGGKGIRVARDEKELETGFRMTEQEA
QTAFGNGGLYMEKFIENFRHIEIQIVGDSYGNVIHLGERDCTIQRRMQKLVEEAPSPILDDETRREMGNAAVRAAKAVNYENAGTIEFIYDLNDNKFYFMEMNTRIQVEHPVTEMVTGIDLVKLQLQVAMGDVLPYKQEDIKLTGHAIEFRINAENPYKNFMPSPGKIEQYLAPGGYGVRIESA
CYTNYTIPPYYDSMVAKLIIHEPTRDEAIMAGIRALSEFVVLGIDTTIPFHIKLLNNDIFRSGKFNTNFLEQNSIMNDEG
Chain B Sequence:
MKKVLIANRGEIAVRIIRACRDLGIQTVAIYSEGDKDALHTQIADEAYCVGPTLSKDSYLNIPNILSIATSTGCDGVHPGYGFLAENADFAELCEACQLKFIGPSYQSIQKMGIKDVAKAEMIKANVPVVPGSDGLMKDVSEAKKIAKKIGYPVIIKATAGGGGKGIRVARDEKELETGFRMTEQE
AQTAFGNGGLYMEKFIENFRHIEIQIVGDSYGNVIHLGERDCTIQRRMQKLVEEAPSPILDDETRREMGNAAVRAAKAVNYENAGTIEFIYDLNDNKFYFMEMNTRIQVEHPVTEMVTGIDLVKLQLQVAMGDVLPYKQEDIKLTGHAIEFRINAENPYKNFMPSPGKIEQYLAPGGYGVRIES
ACYTNYTIPPYYDSMVAKLIIHEPTRDEAIMAGIRALSEFVVLGIDTTIPFHIKLLNNDIFRSGKFNTNFLEQNSIMNDEG
Length of your protein in Amino Acids: 451 amino acids.
Molecular Weight of your protein in kiloDaltons: 50.05 kDA
Molar Extinction coefficient of your protein at 280 nm wavelength: 28685 mol^-1cm-1
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html).
*CDS Gene Sequence (paste as text only):
ATGAAAAAGGTTTTAATTGCAAACCGCGGTGAAATCGCAGTTAGGATTATTCGCGCTTGTCGTGATTTAGGCATCCAAACTGTTGCAATCTATTCTGAAGGGGATAAAGATGCGCTACATACTCAAATTGCTGATGAAGCATATTGCGTAGGTCCCACTTTGTCTAAAGATTCATATTTAAATATTCCGAACATCTTATCT
ATTGCAACTTCTACAGGTTGTGATGGCGTCATCCGGGTTATGGCTTTTTAGCTGAAAATGCTGACTTTGCAGAATTATGCGAAGCATGCCAATTGAAGTTCATTGACCAAGTTATCAATCTATCCAAAAAATGGGTATCAAAGATGTTGCTAAGGCAGAAATGATCAAAGCCAATGTTCCAGTTGTTCCTGGTAGTGAC
GGTTTAATGAAAGACGTCTCAGAAGCTAAGAAAATCGCTAAGAAAATTGGCTATCCGGTCATCATTAAAGCTACTGCTGGTGGTGGCGGAAAAGGTATCCGTGTTGCTCGTGATGAAAAAGAACTTGAAACTGGCTTCCGAATGACAGAACAAGAAGCTCAAACTGCATTTGGTAATGGTGGACTTTATATGGAGA
AATTCATCGAAAACTTCCGCCATATTGAAATCCAAATTGTTGGGGACAGCTATGGTAATGTAATTCATTTAGGAGAACGTGATTGTACAATTCAAAGACGTATGCAGAAATTAGTGGAAGAAGCACCTTCCCCAATTTTAGATGATGAAACACGTCGTGAAATGGGAAATGCCGCAGTTCGTGCAGCGAAAGCTGTA
AATTATGAAAATGCGGGAACAATTGAGTTTATATATGATTTAAATGATAATAAATTTTATTTTATGGAAATGAATACACGTATTCAAGTAGAACATCCTGTAACTGAAATGGTAACAGGAATTGATTTAGTTAAATTACAATTACAAGTTGCTATGGGTGACGTGTTACCGTATAAACAAGAAGATATTAAATAACAGGACA
CGCAATTGAATTTAGAATTAATGCTGAAAATCCTTACAAGAACTTTATGCCATCACCAGGTAAAATTGAGCAATATCTTGCACCAGGTGGATATGGTGTTCGAATAGAGTCAGCATGTTATACTAATTATACGATACCGCCATATTATGATTCGATGGTAGCGAAATTAATCATACATGAACCGACACGAGATGAAGCGAT
TATGGCTGGCATTCGTGCATTAAGTGAATTTGTGGTTCTTGGTATTGATACAACTATTCCATTCCATATTAAATTATTGAATAACGATATATTTAGAAGCGGTAAATTTAATACAAACTTTTTAGAGCAAAATAGCATTATGAATGATGAAGGTTAA
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
*GC% Content for gene:
output
*GC% Content for gene (codon optimized):
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
**